An "off-on" phosphorescent aptasensor for the detection of thrombin based on PRET.

Thrombin plays an important role in the blood coagulation cascade and it stimulates the process of platelet aggregation. Herein, we developed a highly efficient and sensitive phosphorescent aptasensor system for the quantitative analysis of thrombin. The phosphorescence of 3-mercaptopropionic acid capped Mn-doped ZnS quantum dots (MPA-Mn:ZnS QDs) was gradually quenched with the addition of thrombin binding aptamers-BHQ2 (TBA-BHQ2) based on phosphorescence resonance energy transfer (PRET). With the addition of the target analyte thrombin into the system, TBA-BHQ2 could change its spatial structure from a random coil to an antiparallel G-quadruplex which resulted from the combination of thrombin and TBA-BHQ2, leading to the phosphorescence recovery. Finally, the concentration of thrombin could be accurately determined by means of measuring the phosphorescence intensity change value (ΔP). The limit of detection (LOD) was obtained as low as 15.26 pM with wide linear ranges both from 60 to 2000 pM and from 2 to 900 nM. The proposed strategy was also successfully applied for thrombin detection in human serum samples and plasma samples with satisfactory recoveries from 96% to 99% and 95% to 104%, respectively. The long lifetime of phosphorescent QDs possessed a suitable time delay to eliminate autofluorescence and scattered light interference from biological matrices effectively. Thus, the signal to noise ratio of the phosphorescent aptasensor was improved visibly for the analysis of target analytes.

[microRNA-140 suppresses the migration and invasion of colorectal cancer cells through targeting Smad3].

OBJECTIVE: To investigate the effect of microRNA-140 (miR-140) on the migration and invasion of colorectal cancer (CRC) cells and the possible mechanism. METHODS: miR-140 mimics, miR-140 specific inhibitor or small interfering RNA (siRNA) against Smad3 were transfected into human CRC cell line RKO cells respectively, using Oligofectamine or Lipofectamine2000. Quantitative real-time PCR (real-time PCR) was used to measure the expression levels of miR-140 and Smad3 mRNA. Smad3 protein was analyzed by Western blot. The in vitro cell migrating and invasive abilities were determined by wound-healing and Transwell chamber assay after up-regulating or down-regulating miR-140 or knocking down Smad3. RESULTS: The Western blot assays showed that the Smad3 protein level was significantly reduced after up-regulating miR-140 (0.04 ± 0.01), compared with that of (0.47 ± 0.02, P < 0.05) in the control group and that of (0.52 ± 0.06) in the negative control group (P < 0.05 for both). The results of real-time PCR indicated that no significant difference was found in the levels of Smad3 mRNA between miR-140 transfection and NC groups (1.11 ± 0.13 vs. 1.00 ± 0.06, P > 0.05). The wound-healing assay showed that the migrating ability was dramatically attenuated by miR-140 compared with that in the control and NC groups, whereas no significance was found when compared with that of the Smad3 siRNA transfected cells. The number of cells migrating through Transwell chamber without matrigel in the miR-140 group was (76.2 ± 4.4), remarkably lowered than that in the control (267.1 ± 4.9) and NC (336.1 ± 5.7) groups (P < 0.05 for both), but no significant difference between the miR-140 (76.2 ± 4.4) and Smad3 siRNA (83.5 ± 7.3) groups. Transwell chamber with matrigel assay showed that number of cells penetrating through the membrane was (109.5 ± 7.4) in the miR-140 group, significantly lower than that in the control (403.1 ± 5.1) and NC (392.6 ± 8.4) groups (P < 0.05 for both), while Smad3 siRNA transfection had a similar effect (138.8 ± 3.6)(P > 0.05). Down-regulation of miR-140 increased the level of smad3 protein expression, and partially reversed the inhibition of the cell migration and invasion mediated by miR-140. Co-transfection of miR-140 inhibitor and Smad3 siRNA had no significant effect on the Smad3 protein expression and the abilities of cell migration and invasion. CONCLUSIONS: miR-140 regulates the Smad3 expression at the post-transcriptional level. miR-140 suppresses the migrating and invasive abilities of CRC cells, possibly through down-regulation of Smad3. The findings of this study suggest that miR-140 may have a unique potential as a possible biomarker candidate for diagnosis and therapy of tumor metastasis.

Chondroitin sulfate E alleviates ß-amyloid toxicity in transgenic Caenorhabditis elegans by inhibiting its aggregation.

Chondroitin sulfate E (CS-E), which is characterized by oversulfated disaccharide units, has been shown to regulate neuronal adhesion, neurite outgrowth and exert neuroprotective effects. In view of these findings, here we investigated the anti-Alzheimer's disease (AD) activities of CSE by using transgenic Caenorhabditis elegans model of Alzheimer's disease. The behavioral experiments demonstrated that CSE at the concentration of 1 mg/mL significantly delayed the worm paralysis caused by Aß aggregation as compared with control group. Western blot analysis revealed that the level of small oligomers in the transgenic C. elegans was significantly reduced upon treatment with CSE. The number of Aß plaque deposits in transgenic worm was significantly decreased. In addition, CSE also protected the worms from oxidative stress and rescued chemotaxis dysfunction in transgenic strain CL2355. Taken together, these data suggested that CSE could protect against Aß-induced toxicity in C. elegans. These results offer valuable evidence for the future use of CSE in the development of agents for the treatment of AD.

Combined Use of cyclinD1 and Ki67 for Prognosis of Luminal-Like Breast Cancer Patients.

BACKGROUND: Ki67 is a biomarker of proliferation to be used in immunohistochemistry (IHC)-based surrogate assay to determine the necessity of cytotoxic therapy for Luminal-like breast cancer patients. cyclinD1 is another frequently used biomarker of proliferation. A retrospective study was performed here to investigate if these two biomarkers may be combined to improve the prognosis of Luminal-like patients. METHODS: Both Ki67 and cyclinD1 protein levels were measured absolutely and quantitatively using Quantitative Dot Blot method in 143 Luminal-like specimens. Optimized cutoffs for these two biomarkers were developed to evaluate their prognostic roles using Kaplan-Meier overall survival (OS) analysis. RESULTS: cyclinD1 was found as an independent prognostic factor from Ki67 in univariate and multivariate OS analyses. At optimized cutoffs (cyclinD1 at 0.44 µmol/g and Ki67 at 2.31 nmol/g), the subgroup with both biomarkers below the cutoffs (n = 65) had 10-year survival probability at 90% in comparison to those with both biomarkers above the cutoffs (n = 18) with 8-year survival probability at 26% (log-rank test, p <0.0001). This finding was used to modify the surrogate assay using IHC-based cyclinD1 scores, with p-value decreased from 0.031 to 0.00061 or from 0.1 to 0.02, when the Ki67 score of 14 or 20% was used as cutoff, respectively, in the surrogate assay. CONCLUSION: The current study supports the prospective investigation of cyclinD1 relevance in the clinic.

Improving Prognosis of Surrogate Assay for Breast Cancer Patients by Absolute Quantitation of Ki67 Protein Levels Using Quantitative Dot Blot (QDB) Method.

BACKGROUND: Immunohistochemistry (IHC)-based surrogate assay is the prevailing method in daily clinical practice to determine the necessity of chemotherapy for Luminal-like breast cancer patients worldwide. It relies on Ki67 scores to separate Luminal A-like from Luminal B-like breast cancer subtypes. Yet, IHC-based Ki67 assessment is known to be plagued with subjectivity and inconsistency to undermine the performance of the surrogate assay. A novel method needs to be explored to improve the clinical utility of Ki67 in daily clinical practice. MATERIALS AND METHODS: The Ki67 protein levels in a cohort of 253 specimens were assessed with IHC and quantitative dot blot (QDB) methods, respectively, and used to assign these specimens into Luminal A-like and Luminal B-like subtypes accordingly. Their performances were compared with the Kaplan-Meier, univariate, and multivariate survival analyses of the overall survival (OS) of Luminal-like patients. RESULTS: The surrogate assay based on absolutely quantitated Ki67 levels (cutoff at 2.31 nmol/g) subtyped the Luminal-like patients more effectively than that based on Ki67 scores (cutoff at 14%) (Log rank test, p = 0.00052 vs. p = 0.031). It is also correlated better with OS in multivariate survival analysis [hazard ratio (HR) at 6.89 (95% CI: 2.66-17.84, p = 0.0001) vs. 2.14 (95% CI: 0.89-5.11, p = 0.087)]. CONCLUSIONS: Our study showed that the performance of the surrogate assay may be improved significantly by measuring Ki67 levels absolutely, quantitatively, and objectively using the QDB method.

Pulmonary Adenofibroma: Report of a Case with Multiple Masses.

We are reporting one case of multiple pulmonary adenofibromas in a 57-year-old non-smoking female. Ten well-circumscribed masses were identified in both lungs. The masses are characterized by gland-like structures lined by a single layer of simple cuboidal or columnar epithelium. The stroma is abundant and demonstrates compact spindle-cells. The epithelial component is generally positive for CK7, TTF-1, Napsin A. The stromal component displays expression of vimentin, desmin, SMA, h-CALD, ER, PR, Bcl-2, and is negative for CD34, CD117, CD99. We are postulating that the possible histogenesis of these lesions is via proliferation of mesenchymal component of the peribronchial wall, which entraps the epithelium as it expands. Hitherto, this is the first case with multiple lesions reported. Currently, the patient is 11 months post-surgery and doing well.